Abstract:

The present invention relates to methods of screening for modulators of
interleukin 32 (IL-32), to modulators of IL-32 and to their use.

Claims:

1. A method of screening for a modulator of interleukin 32 (IL-32)
activity based on the binding of IL-32 to proteinase 3 (PR-3), which
comprises determining the binding of IL-32 to PR-3 in the presence of a
candidate modulator, comparing the level of said binding to the level of
binding of IL-32 to PR-3 in the absence of said candidate modulator, and
selecting a modulator capable of inhibiting or enhancing said binding.

2. The method according to claim 1, wherein said binding of IL-32 to PR-3
is measured by surface plasmon resonance.

3. The method according to claim 1, wherein said modulator inhibits the
binding of IL-32 to PR-3.

4. The method according to claim 3, wherein said modulator is an inhibitor
of IL-32 activity.

5. The method according to claim 4, wherein said modulator is an inhibitor
of the inflammatory activity of IL-32.

6. The method according to claim 1, wherein said modulator enhances the
binding of IL-32 to PR-3.

7. The method according to claim 6, wherein said modulator is an enhancer
of IL-32 activity.

8. A method of screening for an inhibitor of interleukin 32 (IL-32)
activity based on the proteolytic activity of proteinase 3 (PR-3) on
IL32, which comprises determining the proteolysis of IL-32 by PR-3 in the
presence of a candidate inhibitor and selecting an inhibitor capable of
inhibiting the appearance of an IL-32 fragment generated by the
proteolityc activity of PR-3 or the disappearance of the intact IL-32.

9. The method according to claim 8, wherein said inhibitor inhibits the
inflammatory activity of IL-32.

10. The method according to claim 8, wherein said IL-32 fragment is of
about 16 kDa.

11. The method according to claim 8, wherein said IL-32 fragment is of
about 13 kDa.

12. A method of screening for an inhibitor of interleukin 32 (IL-32)
activity based on the enhancement of IL-32-mediated cytokine secretion by
proteinase 3 (PR-3) in an IL-32 responsive cell, which comprises
contacting IL-32 and PR-3 with an IL-32 responsive cell in the presence
of a candidate inhibitor, determining the concentration of a cytokine in
the culture medium of said cell, comparing to the concentration of said
cytokine in the culture medium of said cell in the absence of said
candidate inhibitor, and selecting for an inhibitor capable of inhibiting
said cytokine secretion.

13. The method according to claim 12, wherein said inhibitor inhibits the
inflammatory activity of IL-32.

14. The method according to claim 12, wherein the IL-32 responsive cell is
a T cell or a macrophage cell.

15. The method according to claim 12, wherein the cytokine is selected
from the group consisting of TNF, IL-8 and MIP-2.

16. A method of screening for a modulator of the activity of interleukin
32 (IL-32) or of the activity of a fragment thereof, which comprises
stimulating an IL-32-responsive cell with IL-32, or with a fragment
thereof in the presence of a candidate modulator, determining the
concentration of a cytokine secreted into the culture medium of said
cell, comparing to the concentration of said cytokine secreted into the
culture medium of said cell in the absence of said candidate modulator
and selecting a modulator capable of inhibiting or enhancing secretion of
said cytokine from said cell.

17. The method according to claim 16, wherein the IL-32 fragment is a
fragment of about 16 kDa generated by the proteolytic activity of PR-3.

18. The method according to claim 16, wherein the IL-32 fragment is a
fragment of about 13 kDa generated by the proteolytic activity of PR-3.

19. The method according to claim 16, wherein the IL-32 responsive cell is
a T cell or a macrophage cell.

20. The method of screening according to claim 16, wherein said candidate
modulator is selected from the group consisting of the inhibitors and
enhancers of claims 8, 9, 14, and 19.

21. The method of screening according to claim 16, wherein the modulator
is an inhibitor of the inflammatory activity of IL-32.

22. A method for treating a disease which is caused or exacerbated by
unregulated production and/or secretion of IL-32 or of a fragment thereof
from cells that express it in a mammal, including a human, which
comprises administering to such mammal in need an effective amount of a
proteinase 3 (PR-3) inhibitor.

23. The method according to claim 22, wherein the disease is caused or
exacerbated by upregulated production and/or secretion of a fragment of
IL-32.

24. The method according to claim 22, wherein the IL-32 fragment is of
about 16 kDa and is generated by the proteolytic activity of PR-3.

25. The method according to claim 22, wherein the IL-32 fragment is of
about 13 kDa and is generated by the proteolytic activity of PR-3.

26. The method according to claim 22, wherein the cells that express IL-32
or a fragment thereof are epithelial cells.

27. The method according to claim 22, wherein the PR-3 inhibitor is
selected from the group consisting of isocoumarin, dichloroisocoumarin,
suramin, hexasulfonated naphtylurea, peptidomimetic agents based on
1,2,5-thiadiazolidin-3-one 1,1-dioxide backbone and their sulfone
derivatives, proteinase inhibitor, elafin, antileukoprotease eglin C,
MNEI (monocyte/neutrophile elastase inhibitor), the bioengineered serpin
LEX032, and a neutralizing anti-PR-3 antibody.

28. A method for treating a disease which is caused or exacerbated by
unregulated production and/or secretion of IL-32 or of a fragment thereof
from cells that express IL-32 in a mammal, including a human, which
comprises administering to such mammal in need an effective amount of an
IL-32 modulator.

29. The method according to claim 28, wherein the disease is caused or
exacerbated by upregulated production and/or secretion of a fragment of
IL-32.

30. The method according to claim 28, wherein the cells that express IL-32
are epithelial cells.

31. The method according to claim 28, wherein the IL-32 fragment is of
about 16 kDa and is generated by the proteolytic activity of PR-3.

32. The method according to claim 28, wherein the IL-32 fragment is of
about 13 kDa and is generated by the proteolytic activity of PR-3.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a Continuation Application of currently pending
U.S. application Ser. No. 11/995,216, which is a National Phase Entry
Application under 35 U.S.C. §371 of co-pending International
Application PCT/IL2006/000798, filed 10 Jul. 2006, which claims benefit
of Israeli application No. 169622, filed on 11 Jul. 2005.

FIELD OF THE INVENTION

[0002]The present invention relates to methods of screening for modulators
of interleukin 32 (IL-32), to modulators of IL-32 and to their use.

BACKGROUND OF THE INVENTION

[0003]Interleukin-32 (IL-32) was initially identified in 1992 by Dahl et
al. as a cytokine-like molecule named natural killer cell transcript 4
(NK4). Since its initial identification in 1992, NK4 has not been
extensively studied and the term NK4 (or NK-4) was assigned also to other
unrelated proteins [Kim, 1989; Smart, 1989; Date, 1997]).

[0004]Increased gene expression of NK4 in peripheral blood mononuclear
cells (PBMC) from patients receiving high-dose IL-2 therapy for malignant
melanoma has been reported, but its function has not been determined
[Panelli, 2002].

[0005]It has been recently reported by Kim et al. (2005) that stimulation
of Raw 264.7 macrophage cells with recombinant NK4 induced secretion of
large amounts of TNF-α in these cells. Therefore, NK4 was
recognized as a pro-inflammatory cytokine and was renamed to IL-32 [Kim,
2005].

[0006]The gene encoding IL-32 resides in the human chromosome 16 p13.3.
The IL-32 gene contains eight exons. Four IL-32 mRNA splice variant
encoding IL-32α, IL-32β, L-32δ and IL-32γ
isoforms, were detected in human natural killer (NK) cells, and the
IL-32γ isoform was identified to be identical to the transcript
previously reported as the NK4 transcript [Kim, 2005]. The IL-32γ
transcript, including exons 3 and 4, encodes a protein isoform having 46
additional amino acids at the N terminus. In the IL-32δ transcript
the second exon is absent and initiation of translation occurs at an ATG
codon present at the third exon. Unlike the other variants, the
IL-32α transcript lacks exons 7 and 8 and encodes a protein isoform
missing 57 amino acid residues at its C terminus. Out of the four IL-32
transcripts, the IL-32α transcript is the most abundant, hence
IL-32α was more extensively characterized. Analysis of the amino
acid sequence of the IL-32α isoform revealed three potential
N-myristoylation sites and one N-glycosylation site [Kim, 2005].

[0007]IL-32α and β were reported to induce secretion of
significant amounts of tumor necrosis factor-α (TNF-α) and
macrophage inflammatory protein-2 (MIP-2) in a dose-dependent manner from
PMA-differentiated THP-1 cells and from mouse Raw cells. Recombinant
IL-32-α and β (rIL-32-α and β) were reported to
induce IL-8 production in non-differentiated human monocytic THP-1 cells.
An Fab fragment of a monoclonal antibody directed against rIL-32β
was found to reduce the biological activity of rIL-32α by up to 70%
in a dose-dependent manner. At the transcriptional level, the 1.2 KB
IL-32 mRNA was detected in several tissues, but was more prominent in
immune cells than in non-immune tissues (Kim, 2005).

[0008]Human peripheral blood mononuclear cells (PBMC), which contain
mostly T cells, produced and secreted IL-32 following stimulation with
Con A (inducing mainly T cell stimulation). Production or secretion of
IL-32 was not detected following lipopolysaccharides stimulation of PBMC
(inducing mainly macrophage stimulation). These results suggest that T
cells are the major producers of IL-32. Nevertheless, stimulation of
epithelial cell lines with IFN-gamma was found to induce IL-32
production.

[0009]The pro inflammatory activity of IL-32 appears to be mediated
through degradation of I-κB, leading to activation of NF-κB.
However, MAP kinase activation by IL-32α has also been reported
[Kim, 2005].

[0010]Proteinase 3 (PR-3, also known as myeloblastin, neutrophil PR-3 and
Wegener autoantigen) is a granule serine protease produced by
neutrophiles/monocytes and is capable of processing multiple biologic
substrates [Baggiolini, 1978; Kao, 1988]. PR-3 degrades a variety of
extracellular matrix proteins, including elastin, fibronectin, type IV
collagen, and laminin and inactivates p65 NF-κB [Preston, 2002].
PR-3 cleaves many pro-hormones and cytokines including angiotensinogen,
TGF-β1, IL-β1, IL-8 and the membrane bound TNF-α into
their active form [Ramaha, 2002; Csernok, 1996; Coeshott, 1999; Padrines,
1994; Robache-Gallea, 1995]. Indeed, high titers of PR-3 autoantibodies
(see below) were found to completely block the cleavage of TNF-α.

[0011]PR-3, which appears both in soluble and cell membrane forms, is the
major autoantigen in Wegener's granulomatosis (WG). Wegener's
granulomatosis is the most common autoimmune necrotizing systemic
vasculitis in adults and is manifested mainly in the respiratory tract
and kidneys [Lamprecht, 2004; Frosch, 2004].

[0012]Autoantibodies to PR-3, known as "Anti-neutrophil cytoplasmic
autoantibodies" (ANCA) are a diagnostic hallmark of WG [van Rossum, 2003;
Jennette, 1997]. The frequency of the membrane PR-3 (mPR-3)-high
phenotype was found to be significantly higher in patients with
ANCA-associated vasculitis and in patients with rheumatoid arthritis.
Hence, membrane PR-3 expression is a risk factor for vasculitis and
rheumatoid arthritis [Witko-Sarsat, 1999]. Expression of PR-3 in the
membranes of neutrophil cells is related to relapse in
PR-3-anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated
vasculitis. Patients with small vessel vasculitis have increased levels
of circulating PR-3 protein in their plasma [Ohlsson, 2003]. High levels
of PR-3 expression on the membrane of neutrophils is also a WG risk
factor and is associated with relapse of WG disease (Rarok A A, Stegeman
C A, Limburg P C, Kallenberg C G. J Am Soc Nephrol. 2002 September;
13(9): 2232-8.

[0013]Apparently, pathogenesis of WG is induced by the binding of ANCA to
PR-3 antigen present on the surface of neutrophils and monocytes. Binding
of ANCA to neutrophils and monocytes causes cell activation, respiratory
burst and release of toxic oxygen radicals and proteolytic enzymes. The
exposure of PR-3 on the cell surface and binding of anti-PR-3
autoantibodies to neutrophils appears to facilitate autoimmunization and
amplification of neutrophil-induced vascular inflammation.

[0014]Gene expression profiles of peripheral mature neutrophils and
monocytes from patients suffering from ANCA diseases manifested in the
kidney showed increased levels of transcripts of a group of genes that
are normally expressed only in bone marrow precursor cells ("left
shift"). PR-3 transcript is included in this group of increased genes and
the increase of PR-3 expression correlated with the disease activity and
with glomerulonephritis [Muller Kobold, 1998; Yang, 2004; Yang, 2002].

[0015]Cystic fibrosis (CF) patients have increased PR-3 mRNA in
circulating monocytes at the time of pulmonary exacerbation [Just, 1999].
Surfactant protein D (SP-D) is an important innate host defense molecule
present in the lung of CF affected patients, which interacts with
CF-associated pathogens [von Bredow, 2003]. SP-D is a target protein for
PR-3. Thus, in CF patients the host defense appears to be impaired due to
proteolysis of SP-D by PR-3, thereby increasing the incidence of
infection of the lung in these patients.

[0016]In patients with inflamed gums functional PR-3 was found to be
expressed in oral epithelial cells and ANCA was found in the patient's
serum. Said epithelial cells expressing functional PR-3 appear to
participate in the inflammatory processes of the gums, including
gingivitis and periodontitis [Uehara, 2004].

[0017]Besides acting on the cell surface and in the extracellular space,
PR-3 enters endothelial cells, where it can mimic caspases, for example,
by cleaving NF-κB and inducing sustained JNK activation. PR-3 also
cleaves and inactivates the major cell cycle inhibitor
p21.sup.Waf1/Cip1/Sdi1. High levels of PR-3 and p21 cleavage product were
found in inflamed human tissue taken from Crohn's disease patients and
from ulcerative colitis [Pendergraft, 2004].

[0018]Dipeptidyl peptidase I (DPPI) is required for the full activation of
neutrophil derived serine proteases such as PR-3. PR-3 knockout mice are
not available, but DPPI-deficient mice were successfully generated
[Adkison, 2002]. The DPPI knockout mice were found to be resistant to
arthritis induction by anti-collagen antibodies and did not accumulated
neutrophils in their joints. Resistance to arthritis induction correlated
with inactivation of neutrophil-derived serine proteases since knockout
mice deficient in serine proteases such as neutrophil elastase
(-/-)×cathepsin G (-/-) were shown to be also resistant to
induction of arthritis by anti-collagen antibodies.

[0019]Enzymatically inactive PR-3 fragments generated by deletion of the
catalytic triad [Yang, 2001] still maintain several biological
activities, including: [0020](i) down-modulation of DNA synthesis in
normal hematopoietic progenitor cells, an effect which can be reversed by
granulocyte-macrophage colony stimulating factor (GM-CSF), implying that
PR-3 can function as a counterbalance to regulators of proliferation
[Skold, 1999]. [0021](ii) Induction of interleukin-8, both at
transcriptional and translational levels [Berger, 1996], and [0022](iii)
Induction of apoptosis in human umbilical vein endothelial cells (HUVEC)
[Yang, 2001].

[0023]As previously mentioned, PR-3 is a serine proteinase and many
well-characterized natural and synthetic serine proteinase inhibitors are
capable of inactivating PR-3, either reversibly or irreversibly. Several
serine proteinase inhibitors were reported to specifically inhibit PR-3.
The synthetic inhibitors 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin
and 3,4-dichloroisocoumarin (DCI) exhibited kI values of 4700 and 2600
M-1s-1, respectively [Kam, 1992]. Suramin, a hexasulfonated
naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of
human neutrophil elastase, cathepsin G, and PR-3. The Ki for PR-3 is
510-7 M [Cadene, 1997]. A general class of peptidomimetic agents
based on 1,2,5-thiadiazolidin-3-one 1,1-dioxide backbone was described
and their sulfone derivatives were found to be time-dependent, potent,
and highly efficient irreversible inhibitors of human leukocyte elastase,
cathepsin G, and PR-3 [Groutas, 1997]. Such compounds were found to be
useful as anti-inflammatory agents (Groutas W C., U.S. Pat. No. 5,550,139
Aug. 27, 1996).

[0024]Other proteinase inhibitors consist of polypeptides of various
sources. Elafin, a human skin derived peptide that inhibits human
leukocyte elastase, was shown to be a potent inhibitor of PR-3, showing
an IC50 of 9.5×10-9 M. Potency was found to be more than
100-fold higher as compared with antileukoprotease and eglin C [Wiedow,
1991; Zani, 2004]. MNEI (monocyte/neutrophile elastase inhibitor) is a 42
kDa serpin superfamily member, which efficiently inhibits proteases with
elastase- and chymotrypsin-like specificities. MNEI rapidly inhibited
PR-3 at a rate >107 M-1s-1 [Cooley, 2001]. A
bioengineered serpin (LEX032) was found to be a time-dependent inhibitor
of PR-3, forming a highly stable enzyme-inhibitor complex (Ki 12 nM)
[Groutas, 1997]. Thus, many serine proteinase inhibitors were
specifically shown to inhibit PR-3.

SUMMARY OF THE INVENTION

[0025]The present invention relates to a method of screening for a
modulator of interleukin 32 (IL-32) activity based on the binding of
IL-32 to PR-3, which comprises determining the binding of IL-32 to PR-3
in the presence of a candidate modulator, comparing the level of said
binding to the level of binding of IL-32 to PR-3 in the absence of said
candidate modulator, and selecting a modulator capable of inhibiting or
enhancing said binding.

[0026]In an embodiment of the invention, said binding of IL-32 to PR-3 is
measured by surface plasmon resonance.

[0027]In one embodiment of the invention, said modulator inhibits the
binding of IL-32 to PR-3.

[0028]In a further embodiment of the invention, the modulator is an
inhibitor of IL-32 activity, preferably an inhibitor of the inflammatory
activity of IL-32.

[0029]In another embodiment of the invention, said modulator enhances the
binding of IL-32 to PR-3 and enhances the activity of IL-32.

[0030]In yet another embodiment of the invention the modulator is a
fragment of PR-3, which binds to IL-32, but is not capable of cleaving
it.

[0031]The present invention provides a method of screening for an
inhibitor of interleukin 32 (IL-32) activity based on the proteolytic
activity of PR-3 on IL32, which comprises determining the proteolysis of
IL-32 by PR-3 in the presence of a candidate inhibitor and selecting an
inhibitor capable of inhibiting the appearance of an IL-32 fragment
generated by the proteolityc activity of PR-3 or the disappearance of the
intact IL-32.

[0032]In one embodiment of the invention, said inhibitor inhibits the
inflammatory activity of IL-32.

[0033]In another embodiment of the invention, said IL-32 fragment is of
about 16 kDa or about 13 kDa.

[0034]In addition the invention provides a method of screening for an
inhibitor of interleukin 32 (IL-32) activity based on the enhancement of
IL-32-mediated cytokine secretion by PR-3 in an IL-32 responsive cell,
which comprises contacting IL-32 and PR-3 with an IL-32 responsive cell
in the presence of a candidate inhibitor, determining the concentration
of a cytokine in the culture medium of said cell, comparing to the
concentration of said cytokine in the culture medium of said cell in the
absence of said candidate inhibitor, and selecting for an inhibitor
capable of inhibiting said cytokine secretion.

[0035]In one embodiment of the invention, said inhibitor inhibits the
inflammatory activity of IL-32.

[0036]In another embodiment of the invention, the IL-32 responsive cell is
a T cell or a macrophage cell.

[0037]In a further embodiment of the invention, the cytokine is selected
from the group consisting of TNF, IL-8 and MIP-2.

[0038]The present invention also provides a method of screening for a
modulator of the activity of interleukin 32 (IL-32) or of the activity of
a fragment thereof, which comprises stimulating an IL-32-responsive cell
with IL-32, or with a fragment thereof in the presence of a candidate
modulator, determining the concentration of a cytokine secreted into the
culture medium of said cell, comparing to the concentration of said
cytokine secreted into the culture medium of said cell in the absence of
said candidate modulator and selecting a modulator capable of inhibiting
or enhancing secretion of said cytokine from said cell.

[0039]In one embodiment of the invention, the IL-32 fragment is the
fragment of about 16 kDa generated by the proteolytic activity of PR-3.

[0040]In another embodiment of the invention, the IL-32 fragment is the
fragment of about 13 kDa generated by the proteolytic activity of PR-3.

[0041]In a further embodiment of the invention, IL-32 responsive cell is a
T cell or a macrophage cell.

[0042]In a still further embodiment of the invention, said candidate
modulator is selected from the group consisting of the inhibitors and
enhancers selected by the methods of the invention.

[0043]In a still further embodiment of the invention, the modulator is an
inhibitor of the inflammatory activity of IL-32.

[0044]The present invention provides modulators of IL-32 activity, such as
inhibitors of IL-32 inflammatory activity, selected by the methods of
screening according to the invention.

[0045]In one embodiment, the invention provides an enhancer of IL-32
activity, selected by the screening methods of the invention.

[0046]In one aspect, the invention provides the use of an inhibitor of
PR-3 in the manufacture of a medicament for the treatment of a disease
which is caused or exacerbated by upregulated production and/or secretion
of IL-32 or of a fragment thereof from cells that express it in a mammal,
including a human.

[0047]In one embodiment of the invention, the disease is caused or
exacerbated by production and/or secretion of a fragment of IL-32.

[0048]In another embodiment of the invention, the IL-32 fragment is of
about 16 kDa and is generated by the proteolytic activity of PR-3.

[0049]In a further embodiment of the invention, the IL-32 fragment is of
about 13 kDa and is generated by the proteolytic activity of PR-3.

[0050]In a still further embodiment of the invention, the cells that
express IL-32 or a fragment thereof are epithelial cells.

[0051]In a yet further embodiment of the invention, the inhibitor of PR-3
is selected from the group consisting of isocoumarin,
dichloroisocoumarin, suramin, hexasulfonated naphtylurea, peptidomimetic
agents based on 1,2,5-thiadiazolidin-3-one 1,1-dioxide backbone and their
sulfone derivatives, proteinase inhibitor, elafin, antileukoprotease
eglin C, MNEI (monocyte/neutrophile elastase inhibitor), the
bioengineered serpin LEX032, and a neutralizing anti-PR-3 antibody.

[0052]In another aspect, the invention teaches the use of an enhancer or
inhibitor of IL-32 screened according to the methods of the invention in
the manufacture of a medicament for the treatment of a disease which is
caused or exacerbated by unregulated production or secretion of IL-32 or
of a fragment thereof from cells that express it in a mammal, including a
human.

[0053]In one embodiment of the invention, the disease is caused or
exacerbated by unregulated production and/or secretion of a fragment of
IL-32.

[0054]In another embodiment of the invention, the IL-32 fragment is of
about 16 kDa and is generated by the proteolytic activity of PR-3.

[0055]In a further embodiment of the invention, the IL-32 fragment is of
about 13 kDa and is generated by the proteolytic activity of PR-3.

[0056]In a yet further embodiment, the modulator is an inhibitor selected
by the screening method of the invention.

[0057]In a yet further embodiment, the production or secretion of IL-32 or
of a fragment thereof from cells that express it is upregulated.

[0058]In a yet further embodiment, the cells that express IL-32 or a
fragment thereof are epithelial cells.

[0059]In a yet further embodiment, the disease is an inflammatory disease.

[0060]In a further aspect, the invention relates to a method for treating
a disease which is caused or exacerbated by upregulated production and/or
secretion of IL-32 or of a fragment thereof from cells that express it in
a mammal, including a human, which comprises administering to such mammal
in need an effective amount of a PR-3 inhibitor.

[0061]In one embodiment of the invention, the disease is caused or
exacerbated by unregulated production and/or secretion of a fragment of
IL-32.

[0062]In another embodiment of the invention, the IL-32 fragment is of
about 16 kDa and is generated by the proteolytic activity of PR-3.

[0063]In a still further embodiment of the invention, the IL-32 fragment
is of about 13 kDa and is generated by the proteolytic activity of PR-3.

[0064]In a still further embodiment of the invention, the cells that
express IL-32 or a fragment thereof are epithelial cells.

[0065]In a still further embodiment of the invention, the PR-3 inhibitor
is selected from the group consisting of isocoumarin,
dichloroisocoumarin, suramin, hexasulfonated naphtylurea, peptidomimetic
agents based on 1,2,5-thiadiazolidin-3-one 1,1-dioxide backbone and their
sulfone derivatives, proteinase inhibitor, elafin, antileukoprotease
eglin C, MNEI (monocyte/neutrophile elastase inhibitor), the
bioengineered serpin LEX032, and a neutralizing anti-PR-3 antibody.

[0066]In a still further aspect, the invention relates to a method for
treating a disease which is caused or exacerbated by unregulated
production and/or secretion of IL-32 or of a fragment thereof from cells
that express it in a mammal, including a human, which comprises
administering to such mammal in need an effective amount of a modulator
selected according to any one of the screening methods of the invention.

[0067]In one embodiment of the invention, the disease is caused or
exacerbated by upregulated production and/or secretion of a fragment of
IL-32.

[0068]In another embodiment of the invention, the cells that express IL-32
are epithelial cells.

[0069]In a still further embodiment of the invention, the IL-32 fragment
is of about 16 kDa or 13 kDa and is generated by the proteolytic activity
of PR-3.

[0070]The invention also provides a polypeptide fragment of IL-32,
obtained by the proteolysis of IL-32 by PR-3, such as the IL-32 fragment
consisting of about 16 kDa or of about 13 kDa or, a mutein, fusion
protein, functional derivative, a circularly permuted derivative, or
active fraction thereof.

[0071]In addition, the invention provides a pharmaceutical composition
comprising a polypeptide fragment of IL-32 obtained by the proteolysis of
IL-32 by PR-3, such as the IL-32 fragment consisting of about 16 kDa or
the IL-32 fragment consisting of about 13 kDa or, a mutein, fusion
protein, functional derivative, a circularly permuted derivative, or
active fraction thereof and a pharmaceutically acceptable carrier.

[0075]FIG. 1 shows fractions of urinary proteins eluted from the IL-32
immobilized affinity chromatography column resolved in SDS-PAGE. IL-32
was immobilized on an Affigel 15 resin and concentrated urinary proteins
from 500 L of urine were passed on the resin; the resin was washed and
resin-bound proteins were eluted by a pH 2.2 buffer in 1 ml fractions.
Aliquots (60 μl) from the various fractions were resolved in SDS-PAGE
(12% acrylamide; non-reducing conditions) and the gel was silver-stained.
Lane 1 is the wash fraction; lanes 2-6 are the elution fractions 1 to 5,
respectively and lane 7 shows the molecular mass markers, indicated on
the right side (in kDa). The arrow in the figure shows an IL-32-binding
protein of 30±2 kDa that eluted mainly in fraction 3 and was
identified as PR-3.

[0076]FIG. 2 A-B shows kinetics of human urinary PR-3 binding to human
IL-32 as measured by surface plasmon resonance. FIG. 2 A. IL-32 (20
μg/ml) in acetate buffer pH 4.6) was immobilized to a single channel
of a BIAcore chip as recommended by the manufacturer (Amersham Pharmacia,
Uppsala Sweden). Aliquots of elution fraction 3 from the IL-32 affinity
column were brought to a concentration of 10, 20, 30, 40 and 80 nM
(binding curves from bottom to top, respectively) and analyzed by the
BIAcore system. The binding data gave a kD of 2.65×10-8 M.
FIG. 2 B. The same analysis was done with PR-3 that was inactivated by
phenyl methyl sulfonyl fluoride (PMSF). The resulting kD was
7.9×10-8 M.

[0077]FIG. 3 A-B shows kinetics of commercially available human PR-3
binding to human IL-32 as measured by surface plasmon resonance. For
details see FIG. 2. The binding data gave a kD of 1.2×10-8 M
with active PR-3, which considerably degraded the immobilized IL-32 (FIG.
3 A). The kD of commercially available PR-3 that was inactivated by PMSF
(FIG. 3 B) was 3.5×10-8 M.

[0078]FIG. 4 shows the kinetics of digestion of 125I-IL-32 by urinary
PR-3. Lane 1 shows the resolved undigested 125I-IL-32, Lane 5 the
molecular weight markers (in kDa see the right side of the Fig.) and
lanes 2-4 and 6-7 show 125I-IL-32 following incubation with PR-3 for
1, 5, 15, 30 and 60 minutes at 37° C., respectively. After 1
minute of incubation with PR-3 the 20 kDa IL-32 (Lane 1) was cleaved into
two cleavage products of 16 and 13 kDa (Lane 2). After 5 min. (Lane 3)
the band corresponding to the intact IL-32 disappeared and the cleavage
products remained stable for at least 60 min. (lanes 3, 4, 6, and 7). The
resulting 125I-IL-32 fragments of apparent MW of 20 kDa, 16 kDa and
13 kDa are indicated on the left side of the FIG.

[0079]FIG. 5 shows the comparison of IL-32 cleavage by urinary and
commercial PR-3. The experiment was carried out essentially as indicated
in FIG. 4 (Example 5) except, that incubation of PR-3 and IL32 was
limited to up to 5 minutes and that both urinary and commercial PR-3 were
used. Urinary (Lines 1-3) or commercial PR-3 (Lines 5-7) were incubated
with 125I-IL-32 for 0, 1 and 5 minutes. The results show that
commercial PR-3 was more efficient than the urine-derived PR-3 since the
20 kDa IL-32 was completely cleaved into its 16 and 13 kDa fragments
after 1 min (compare lanes 6 and 2).

[0080]FIG. 6 shows that phenyl methyl sulfonyl fluoride (PMSF), a serine
protease inhibitor, blocks the enzymatic activity of both urinary and
commercial PR3 and blocks the cleavage of IL-32. Lanes 1-4 show
125I-IL-32 following incubation with urinary PR-3 for 1 and 5 min.,
either not treated (Lanes 1 and 2) or pre-treated with PMSF (Lanes 3 and
4). Lanes 7-10 show 125I-IL-32 following incubation with commercial
PR-3 for 1 and 5 min., either not treated (Lanes 9 and 10) or pre-treated
with PMSF (Lanes 7 and 8). Molecular weight markers (in kDa; lane 5) are
indicated at the right side. Lane 6 shows undigested 125I-IL-32. The
figure shows the extent of cleavage of IL-32 by urinary and commercial
PR-3 after 1 and 5 min. of incubation (lanes 1 and 5 for urinary and 9
and 10 for commercial) and that PMSF completely blocked IL-32 cleavage by
both commercial and urinary PR-3.

[0081]FIG. 7 shows enhanced biological activity of IL-32 pre-treated with
PR-3 in mouse Raw 264.7 cells. Mouse macrophage Raw 264.7 cell line were
seeded in a 96 well plate and incubated with IL-32 (20 or 200 ng/ml),
IL-32 pretreated with PR-3 (100 ng/ml) for 5 min. or with PR-3 alone as
the a control, and MIP-2 secreted into the cell culture medium was
measured. The results summarized in the Fig. show that IL-32 induced the
secretion of MIP-2 by Raw 264.7 cells and that preincubation of IL-32
with PR-3 for 5 minutes prior to treatment of the cells (shown in FIGS.
4-6 to cleave the 20 kDa IL-32 into 16 and 13 kDa fragments) enhanced
MIP-2 secretion. PR-3 alone had no effect on the cells.

[0082]FIG. 8 shows enhanced biological activity of IL-32 pre-treated with
PR-3 in human peripheral blood mononuclear cells (PBMC). Human PBMC cells
were seeded in 96 well plates and incubated with IL-32 (20 or 200 ng/ml),
IL-32 pretreated with PR-3 (100 ng/ml) for 5 min. or with PR-3 alone as
the a control. The content of IL-8 into the cell culture medium was
measured. The results show that IL-32 induced the secretion of IL-8 in
human PBMC cells and that incubation of IL-32 for 5 minutes with PR-3
(shown to induce complete IL-32 degradation in FIGS. 4-6) prior to cell
treatment enhanced IL-8 secretion. Incubation of the cells with PR-3
alone did not induce IL-8 in the mouse cells.

DETAILED DESCRIPTION OF THE INVENTION

[0083]The present invention relates to methods of screening (or
identification or selection) for a modulator of the activity of
interleukin 32 (IL-32), based on our findings that PR-3 binds IL-32
causes IL-32 proteolysis and the proteolytic fragments generated by the
action of PR-3 have enhanced activity.

[0084]In addition, the present invention relates to the modulators of
IL-32 activity identified by said method of screening and to the use of
said modulators in a disease which is caused or exacerbated by the
unregulated production and/or secretion of IL-32 or of a fragment
thereof.

[0085]The invention also relates to inhibitors of IL-32 activity which are
fragments of PR-3 which bind to, but are incapable of cleaving, IL-32.

[0086]Cytokines normally serve to enhance defense. However, when acting in
excess, they may cause great damage, not lesser than that which pathogens
can cause. In fact, in many diseases unwarranted effects of cytokines
constitute a major pathogenic cause. Upregulated production and/or
secretion of IL-32 or a fragment thereof may cause an inflammatory
disease or may exacerbate an inflammatory disease. An inhibitor of IL-32
activity is desired to inhibit inflammatory diseases caused or
exacerbated by upregulation of IL-32 production and/or secretion.
Down-regulated production and/or secretion of IL-32 or a fragment thereof
may impair host defense, thereby increasing the incidence of infections
and cancer. An enhancer of IL-32 activity is desired to inhibit
infections and cancer caused or exacerbated by downregulation of IL-32
production and/or secretion. The present invention is based on our
findings that PR-3 binds to IL-32 with a high affinity and degrades IL-32
into 13 and 16 kDa fragments, which exhibit enhanced biological activity
as compared to the biological activity of intact IL-32.

[0087]As is illustrated in the Examples section which follows, we have
established that PR-3 plays a crucial role in activation of IL-32.

[0088]Briefly, we concentrated and loaded on a column consisting of human
IL-32 bound to agarose, crude human urinary proteins. We eluted the
column-bound proteins by lowering the column pH and analyzed the eluted
proteins by SDS-PAGE (10% acrylamide). A protein band of 28-32 kDa
further identified as the neutrophil-derived serine protease PR-3 (as
described in Example 1 below) was specifically enriched in one of the
eluted fractions (FIG. 1 elution fraction 3).

[0089]We found that PR-3 binds to IL-32 with high affinity (Kd of about
2.65×10-8M with the urinary PR-3 and Kd of about
1.20×10-8 M with the commercial PR-3, see Example 2). The
binding affinity of PR-3 to IL-32 decreased very little after
pre-incubating PR-3 with phenyl methyl sulfonyl fluoride (PMSF),
indicating that binding of IL-32 to PR-3 is not dependent on the
enzymatic activity of the latter.

[0093]We showed that the IL-32 protein (of about 20 kDa) disappeared after
one min incubation with commercial PR-3, concomitantly with the
generation of the 13 kDa (SEQ ID NO: 1) and 16 kDa (SEQ ID NO: 2)
fragments. Pretreatment of PR-3 with PMSF completely blocked the cleavage
of IL-32.

[0094]To our surprise we found that the proteolytic fragments of IL-32, of
13 kDa (SEQ ID NO: 1) and 16 kDa (SEQ ID NO: 2), had enhanced biological
activity compared to the intact IL-32 protein (as demonstrated in Example
8). The cleavage of IL-32 and the consequent enhancement of the
biological activity were both blocked by the action of PMSF.

[0095]For the purpose of the present description the expression
"biological activity of IL-32" refers inter alia to at least one of the
following biological properties:

(i) induction of MIP-2, (ii) induction of TNF, and (iii) induction of
IL-8.

[0096]Therefore, based on our results, modulating the binding of IL-32 to
PR-3 (or formation of the IL-32-PR-3 complex) will modulate, namely
enhance or inhibit, the activity of IL-32. As used herein, the expression
"inhibiting the activity of IL-32" means the capability of an inhibitor
to inhibit any IL-32 activity in addition to blocking, e.g. partial
inhibition, or the like.

[0097]The invention provides a method of identifying a modulator molecule,
the method comprising identifying a molecule capable of enhancing or
inhibiting the binding of PR-3 to IL-32 (or PR-3-IL-3 complex formation),
said molecule being the modulator.

[0098]In one embodiment, the present invention relates to a method of
screening for a modulator of interleukin 32 (IL-32) activity based on the
binding of IL-32 to PR-3, which comprises determining the binding of
IL-32 to PR-3 in the presence of a candidate modulator, comparing to the
binding of IL-32 to PR-3 in the absence of said candidate modulator, and
selecting (or identifying) a modulator capable of inhibiting or enhancing
said binding.

[0099]In one embodiment the candidate modulator is an organic molecule
which may be designed by combinatorial chemistry.

[0100]The term "IL-32" according to the invention, includes all the IL-32
isoforms such as IL-32 α, IL-32 β, IL-32 γ and IL-32
δ.

[0101]The term "modulator of IL-32 activity" means an inhibitor or an
enhancer of IL-32 activity.

[0102]As used herein, the expression "binding to IL-32" means the
capability of PR-3 to bind IL-32, e.g. as evidenced by PR-3 binding to
IL32 when affinity purified as in Example 1 or when tested in BIAcore as
in Example 3.

[0103]As mentioned, cytokines normally serve to enhance defense. However,
when acting in excess, they may cause great damage, not lesser than that
which pathogens can cause. In fact, in many diseases unwarranted effects
of cytokines constitute a major pathogenic cause. IL-32 acting in excess
may cause excessive function of TNF and inflammation. In a further
embodiment of the present invention, the modulator is an inhibitor of the
inflammatory activity of IL-32.

[0104]Deficiency of IL-32 may impair host defense, thereby increasing the
incidence of infections and cancer. In a further embodiment of the
present invention, the modulator is an enhancer of the activity of IL-32.

[0105]Based on our results, inhibiting proteolysis of IL-32 by PR-3 will
result in the inhibition of the activity of IL-32 (As exemplified below
with PMSF).

[0106]The invention provides a method of identifying a modulator molecule,
the method comprising identifying a molecule capable of enhancing or
inhibiting the proteolysis of IL-32 by PR-3, said molecule being the
modulator.

[0107]In one embodiment the invention relates to a method of screening for
an inhibitor of IL-32 (IL-32) activity based on the proteolytic activity
of PR-3 on IL-32, which comprises determining the proteolysis of IL-32 by
PR-3 in the presence of a candidate inhibitor and selecting an inhibitor
capable of inhibiting the appearance of an IL-32 fragment generated by
the proteolityc activity of PR-3 or the disappearance of the intact
IL-32.

[0108]In a further embodiment, determining the proteolysis of IL-32 is
carried out by monitoring IL-32 fragments generated by PR-3, having
molecular weights of about 16 and 13 kDa and/or by monitoring the
disappearance of the intact IL-32 of molecular weight of about 20 kDa as
exemplified in Examples 5-7.

[0110]In accordance with another embodiment of the present invention,
fragments of PR-3, which can be obtained by cleavage of PR-3 by any known
method, which bind IL-32, but are not capable of cleaving it, may be
employed to abolish the enhancing function of PR-3 on the biological
activity of IL-32 as manifested by the cleaved IL-32.

[0111]In accordance with the invention PR-3 is cleaved by CNBr, or trypsin
and the resulting fragments of PR-3 are checked for affinity to IL-32 by
BIACORE and compared to the affinity of intact PR-3. Fragments with high
affinity are then further tested for inhibition of the biological
activity of IL-32.

[0112]The invention provides a method of identifying a modulator molecule,
the method comprising identifying a molecule capable of enhancing or
inhibiting the biological activity of IL-32 enhanced by PR-3, wherein the
biological activity is manifested by secretion of a cytokine such as
IL-8, TNF and MIP-4 by IL-32 responsive cells, said molecule being the
modulator.

[0113]In one embodiment, the invention relates to a method of screening
for an inhibitor of interleukin 32 (IL-32) activity based on the
enhancement of IL-32-mediated cytokine secretion by PR-3 in an IL-32
responsive cell, which comprises contacting IL-32 and PR-3 with an IL-32
responsive cell in the presence of a candidate inhibitor, determining the
concentration of a cytokine in the culture medium of said cell, comparing
to the concentration of said cytokine in the culture medium of said cell
in the absence of said candidate inhibitor, and selecting for an
inhibitor capable of inhibiting said cytokine secretion.

[0114]The invention provides a method of identifying a modulator molecule,
the method comprising identifying a molecule capable of enhancing or
inhibiting the activity of IL-32 or a molecule capable of enhancing or
inhibiting the activity or the production of IL-32 13 kDa (SEQ ID NO: 1)
and 16 kDa (SEQ ID NO: 2) fragments, said molecule being the modulator.

[0115]In one embodiment, the present invention relates to a method of
screening for a modulator of the activity of interleukin 32 (IL-32) or a
molecule capable of enhancing or inhibiting the activity or production of
fragment thereof, which comprises stimulating an IL-32-responsive cell
with IL-32, or with a fragment thereof in the presence of a candidate
modulator, determining the concentration of a cytokine secreted into the
culture medium of said cell, comparing to the concentration of said
cytokine secreted into the culture medium of said cell in the absence of
said candidate modulator and selecting a modulator capable of inhibiting
or enhancing secretion of said cytokine from said cell.

[0116]As used herein, the expression "enhancement of IL-32-mediated
cytokine secretion by PR-3 "means the capability of PR-3 to enhance IL-32
mediated cytokine secretion in an IL-32 responsive cell, for example, as
evidenced by enhancement of IL-8 or MIP-2 secretion stimulated by IL32
incubated with PR-3 (as exemplified in Example 8).

[0117]In one embodiment, the present invention relates to modulators of
IL-32 found by the method of the present invention and to their use in
the manufacture of a medicament for the treatment of a disease which is
caused or exacerbated by unregulated production or secretion of IL-32 or
of a fragment thereof from cells that express it in a mammal, including a
human.

[0118]In a further embodiment, the present invention relates to the use of
a modulator of PR-3 in the manufacture of a medicament for the treatment
of a disease, which is caused or exacerbated by unregulated production
and/or secretion of IL-32 or of a fragment thereof from cells that
express it in a mammal, including a human.

[0119]Our results show that inhibitors of the present invention, such as
inhibitors of PR-3, inhibitors of binding of PR-3 to IL-32, inhibitors of
IL-32 proteolysis by PR-3 (exemplified below with PMSF) and inhibitors of
the biological activity of IL-32 identified by the methods of screening
of the present invention may find use as inhibitors of IL-32 activity for
example, in diseases in which endogenous production or exogenous
administration of IL-32 is the cause of the disease or exacerbates the
situation of the patient.

[0120]In one embodiment of the invention, an inhibitor of IL-32 activity
identified by the method of screening of the present invention, is used
in a disease, which is caused or exacerbated, by the increased production
and/or secretion of IL-32 such as inflammation.

[0122]The present invention provides methods for treating a disease which
is caused or exacerbated by unregulated production and/or secretion of
IL-32 or of a fragment thereof in a cell that express it, such as T cells
and endothelial cells, in a mammal, including a human, which comprises
administering to such mammal in need an effective amount of PR-3
inhibitor.

[0123]The term "treatment" or "treating" is intended to include the
administration of the compound of the invention to a subject for purposes
which may include prophylaxis, amelioration, prevention or cure of caused
or exacerbated by unregulated IL-32 activity. Such treatment need not
necessarily completely ameliorate the inflammatory response or other
responses related to the specific disorder. Further, such treatment may
be used as sole treatment or in conjunction with other traditional
treatments for reducing the deleterious effects of the disease, disorder
or condition as known to those of skill in the art.

[0124]The methods of the invention may be provided as a "preventive"
treatment before detection of, for example, an inflammatory state, so as
to prevent the disorder from developing in patients at high risk for the
same, such as, for example, transplant patients.

[0125]The term "cancer" refers to various cancer-associated conditions
including metastasis, tumor growth, and angiogenesis.

[0127]The invention also includes the use of neutralizing antibodies
against PR-3 as well as against their muteins, fused proteins, salts,
functional derivatives and active fractions. The term "antibody" is meant
to include polyclonal antibodies, monoclonal antibodies (MAbs), chimeric
antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be
labeled in soluble or bound form, and humanized antibodies as well as
fragments thereof provided by any known technique, such as, but not
limited to enzymatic cleavage, peptide synthesis or recombinant
techniques.

[0129]In one aspect, the invention relates to a polypeptide fragment of
IL-32 of about 16 kDa (SEQ ID NO: 2) and to a polypeptide fragment of
IL-32 of about 13 kDa (SEQ ID NO: 1), both active and obtained by the
proteolytic activity of PR-3.

[0132]As used herein the term "muteins" refers to analogs of a protein, in
which one or more of the amino acid residues of the naturally occurring
components of the protein are replaced by different amino acid residues,
or are deleted, or one or more amino acid residues are added to the
original sequence of the protein, without changing considerably the
activity of the resulting products as compared with the original protein.
These muteins are prepared by known synthesis and/or by site-directed
mutagenesis techniques, or any other known technique suitable therefore.

[0133]Muteins in accordance with the present invention include proteins
encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or
RNA, which encodes the protein, in accordance with the present invention,
under stringent conditions. The term "stringent conditions" refers to
hybridization and subsequent washing conditions, which those of ordinary
skill in the art conventionally refer to as "stringent". See Ausubel et
al., Current Protocols in Molecular Biology, supra, Interscience, N.Y.,
§§6.3 and 6.4 (1987, 1992), and Sambrook et al. (Sambrook, J.
C., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).

[0134]Without limitation, examples of stringent conditions include washing
conditions 12° 20-° C. below the calculated Tm of the
hybrid under study in, e.g., 2×SSC and 0.5% SDS for 5 minutes,
2×SSC and 0.1% SDS for 15 minutes; 0.1×SSC and 0.5% SDS at
37° C. for 30-60 minutes and then, a 0.1×SSC and 0.5% SDS at
68° C. for 30-60 minutes. Those of ordinary skill in this art
understand that stringency conditions also depend on the length of the
DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed
oligonucleotide probes. If mixed probes are used, it is preferable to use
tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.

[0135]Any such mutein preferably has a sequence of amino acids
sufficiently duplicative of that of the polypeptides of the invention,
such as to have substantially similar, or even better, activity to the
polypeptides of the invention. For example, one characteristic activity
of IL-32 is its capability of inducing secretion of TNF in responsive
cells. An ELISA type assay for measuring the binding of TNF are described
in the art. As long as the mutein has substantial activity of the
polypeptide of the invention, it can be considered to have substantially
similar activity to the polypeptide of the invention. Thus, it can be
determined whether any given mutein has at least substantially the same
activity as the polypeptide of the invention by means of routine
experimentation comprising subjecting such a mutein, e.g., to simple
assays to determine whether or not it induces secretion of a cytokine
such as TNF, IL-8 or MIP-2 in IL-32 responsive cells.

[0136]In a preferred embodiment, any such mutein has at least 40% identity
or homology with the amino acid sequence of the polypeptide of the
invention. More preferably, it has at least 50%, at least 60%, at least
70%, at least 80% or, most preferably, at least 90% identity or homology
thereto.

[0137]Identity reflects a relationship between two or more polypeptide
sequences or two or more polynucleotide sequences, determined by
comparing the sequences. In general, identity refers to an exact
nucleotide to nucleotide or amino cid to amino acid correspondence of the
two polynucleotides or two polypeptide sequences, respectively, over the
length of the sequences being compared.

[0138]For sequences where there is not an exact correspondence, a "percent
identity" may be determined. In general, the two sequences to be compared
are aligned to give a maximum correlation between the sequences. This may
include inserting "gaps" in either one or both sequences, to enhance the
degree of alignment. A percent identity may be determined over the whole
length of each of the sequences being compared (so-called global
alignment), that is particularly suitable for sequences of the same or
very similar length, or over shorter, defined lengths (so-called local
alignment), that is more suitable for sequences of unequal length.

[0139]Methods for comparing the identity and homology of two or more
sequences are well known in the art. Thus for instance, programs
available in the Wisconsin Sequence Analysis Package, version 9.1
(Devereux J et al 1984, Nucleic Acids Res. 1984 Jan. 11; 12(1 Pt 1):
387-95.), for example the programs BESTFIT and GAP, may be used to
determine the % identity between two polynucleotides and the % identity
and the % homology between two polypeptide sequences. BESTFIT uses the
"local homology" algorithm of Smith and Waterman (J Theor Biol. 1981 Jul.
21; 91(2): 379-80 and J Mol. Biol. 1981 Mar. 25; 147(1): 195-7. 1981) and
finds the best single region of similarity between two sequences. Other
programs for determining identity and/or similarity between sequences are
also known in the art, for instance the BLAST family of programs
(Altschul S F et al, 1990 J Mol. Biol. 1990 Oct. 5; 215(3): 403-10, Proc
Natl Acad Sci USA. 1990 July; 87(14): 5509-13, Altschul S F et al,
Nucleic Acids Res. 1997 Sep. 1; 25(17): 3389-402, accessible through the
home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R,
Methods Enzymol. 1990; 183:63-98. Pearson J Mol. Biol. 1998 Feb. 13;
276(1): 71-84).

[0140]Muteins of the polypeptide of the invention, which can be used in
accordance with the present invention, or nucleic acid coding therefore,
include a finite set of substantially corresponding sequences as
substitution peptides or polynucleotides which can be routinely obtained
by one of ordinary skill in the art, without undue experimentation, based
on the teachings and guidance presented herein.

[0141]Preferred changes for muteins in accordance with the present
invention are what are known as "conservative" substitutions.
Conservative amino acid substitutions of the polypeptide of the invention
may include synonymous amino acids within a group which have sufficiently
similar physicochemical properties that substitution between members of
the group will preserve the biological function of the molecule (Grantham
Science. 1974 Sep. 6; 185(4154): 862-4). It is clear that insertions and
deletions of amino acids may also be made in the above-defined sequences
without altering their function, particularly if the insertions or
deletions only involve a few amino acids, e.g., under thirty, and
preferably under ten, and do not remove or displace amino acids which are
critical to a functional conformation, e.g., cysteine residues. Proteins
and muteins produced by such deletions and/or insertions come within the
purview of the present invention.

[0142]Preferably, the synonymous amino acid groups are those defined in
Table 1. More preferably, the synonymous amino acid groups are those
defined in Table 2; and most preferably the synonymous amino acid groups
are those defined in Table 3.

[0143]Examples of production of amino acid substitutions in proteins which
can be used for obtaining muteins of the polypeptide of the invention,
for use in the present invention include any known method steps, such as
presented in U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Mark
et al; 5,116,943 to Koths et al., 4,965,195 to Namen et al; 4,879,111 to
Chong et al; and 5,017,691 to Lee et al; and lysine substituted proteins
presented in U.S. Pat. No. 4,904,584 (Shaw et al).

[0144]"Functional derivatives" as used herein cover derivatives of the
polypeptide of the invention, and their muteins, which may be prepared
from the functional groups which occur as side chains on the residues or
are additions to the N- or C-terminal groups, by means known in the art,
and are included in the invention as long as they remain pharmaceutically
acceptable, i.e. they do not destroy the activity of the protein which is
substantially similar to the activity of the polypeptide of the
invention, and do not confer toxic properties on compositions containing
it.

[0145]"Functional derivatives" also comprise multimers made up of the
polypeptide of the invention in which changes have been introduced in the
sequence of the amino acids making up the polypeptide of the invention by
any conventional method. These changes may comprise elongation or
truncation of the polypeptide of the invention or deletion or replacement
of one or more amino acids making up the polypeptide of the invention. It
is understood that none of the above changes may affect the properties of
the polypeptide of the invention.

[0146]These derivatives may, for example, include polyethylene glycol
side-chains, which may mask antigenic sites and extend the residence of
the polypeptide of the invention in body fluids. Other derivatives
include aliphatic esters of the carboxyl groups, amides of the carboxyl
groups by reaction with ammonia or with primary or secondary amines,
N-acyl derivatives of free amino groups of the amino acid residues formed
with acyl moieties (e.g. alkanoyl or carboxylic aroyl groups) or O-acyl
derivatives of free hydroxyl groups (for example that of seryl or
threonyl residues) formed with acyl moieties.

[0147]An "active fraction" according to the present invention may e.g. be
a fragment of the 13 kDa or 16 kDa fragments. The term fragment refers to
any subset of the molecule, that is, a shorter peptide that retains the
desired biological activity e.g. inducing TNF secretion by IL-32
responsive cells. Fragments may readily be prepared by removing amino
acids from either end of the polypeptide of the invention and testing the
resultant fragment for its biological properties. Proteases for removing
one amino acid at a time from either the N-terminal or the C-terminal of
a polypeptide are known, and so determining fragments, which retain the
desired biological activity, involves only routine experimentation.

[0148]As active fractions of the polypeptide of the invention, muteins and
fused proteins thereof, the present invention further covers any fragment
of the polypeptide chain of the protein molecule alone or together with
associated molecules or residues linked thereto, e.g., sugar or phosphate
residues, or aggregates of the protein molecule or the sugar residues by
themselves, provided said fraction has substantially similar activity to
the polypeptide of the invention.

[0149]In yet a further embodiment, the substance according to the
invention comprises an immunoglobulin fusion, i.e. the molecules
according to the invention are fused to all or a portion of an
immunoglobulin. Methods for making immunoglobulin fusion proteins are
well known in the art, such as the ones described in WO 01/03737, for
example. The person skilled in the art will understand that the resulting
fusion protein of the invention retains the biological activity of the
polypeptide of the invention. The resulting fusion protein ideally has
improved properties, such as an extended residence time in body fluids
(half-life), increased specific activity, increased expression level, or
facilitated purification of the fusion protein.

[0150]Preferably, the substance according to the invention is fused to the
constant region of an Ig molecule. It may be fused to heavy chain
regions, like the CH2 and CH3 domains of human IgG1, for example. Other
isoforms of Ig molecules are also suitable for the generation of fusion
proteins according to the present invention, such as isoforms IgG2 or
IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins
may be monomeric or multimeric, hetero- or homomultimeric.

[0151]The term "salts" herein refers to both salts of carboxyl groups and
to acid addition salts of amino groups of the polypeptide of the
invention or analogs thereof. Salts of a carboxyl group may be formed by
means known in the art and include inorganic salts, for example, sodium,
calcium, ammonium, ferric or zinc salts, and the like, and salts with
organic bases as those formed, for example, with amines, such as
triethanolamine, arginine or lysine, piperidine, procaine and the like.
Acid addition salts include, for example, salts with mineral acids, such
as, for example, hydrochloric acid or sulfuric acid, and salts with
organic acids, such as, for example, acetic acid or oxalic acid. Of
course, any such salts must retain the biological activity of the
polypeptide of the invention such as the ability to induce TNF, IL-8 or
MIP-2 in IL-32 responsive cells.

[0152]The term "circularly permuted" as used herein refers to a linear
molecule in which the termini have been joined together, either directly
or through a linker, to produce a circular molecule, and then the
circular molecule is opened at another location to produce a new linear
molecule with termini different from the termini in the original
molecule. Circular permutations include those molecules whose structure
is equivalent to a molecule that has been circularized and then opened.
Thus, a circularly permuted molecule may be synthesized de novo as a
linear molecule and never go through a circularization and opening step.
The particular circular permutation of a molecule is designated by
brackets containing the amino acid residues between which the peptide
bond is eliminated. Circularly permuted molecules, which may include DNA,
RNA and protein, are single-chain molecules, which have their normal
termini fused, often with a linker, and contain new termini at another
position. See Goldenberg, et al. J. Mol. Biol., 165: 407-413 (1983) and
Pan et al. Gene 125: 111-114 (1993), both incorporated by reference
herein. Circular permutation is functionally equivalent to taking a
straight-chain molecule, fusing the ends to form a circular molecule, and
then cutting the circular molecule at a different location to form a new
straight chain molecule with different termini. Circular permutation thus
has the effect of essentially preserving the sequence and identity of the
amino acids of a protein while generating new termini at different
locations.

[0153]The invention provides a pharmaceutical composition comprising the
polypeptide fragment of IL-32 of about 16 kDa or of about 13 kDa and a
pharmaceutically acceptable carrier.

[0154]The invention provides also a pharmaceutical composition comprising
the polypeptide fragment of IL-32 of about 16 kDa or of about 13 kDa and
a pharmaceutically acceptable carrier for the treatment of a disease
induced by a pathogen.

[0155]The definition of "pharmaceutically acceptable" is meant to
encompass any carrier, which does not interfere with effectiveness of the
biological activity of the active ingredient and that is not toxic to the
host to which it is administered. For example, for parenteral
administration, the active protein(s) may be formulated in a unit dosage
form for injection in vehicles such as saline, dextrose solution, serum
albumin and Ringer's solution.

[0156]The active ingredients of the pharmaceutical composition according
to the invention can be administered to an individual in a variety of
ways. The routes of administration include intradermal, transdermal (e.g.
in slow release formulations), intramuscular, intraperitoneal,
intravenous, subcutaneous, oral, intracranial, epidural, topical, and
intranasal routes. Any other therapeutically efficacious route of
administration can be used, for example absorption through epithelial or
endothelial tissues or by gene therapy wherein a DNA molecule encoding
the active agent is administered to the patient (e.g. via a vector),
which causes the active agent to be expressed and secreted in vivo. In
addition, the protein(s) according to the invention can be administered
together with other components of biologically active agents such as
pharmaceutically acceptable surfactants, excipients, carriers, diluents
and vehicles.

[0157]For parenteral (e.g. intravenous, subcutaneous, intramuscular)
administration, the active protein(s) can be formulated as a solution,
suspension, emulsion or lyophilized powder in association with a
pharmaceutically acceptable parenteral vehicle (e.g. water, saline,
dextrose solution) and additives that maintain isotonicity (e.g.
mannitol) or chemical stability (e.g. preservatives and buffers). The
formulation is sterilized by commonly used techniques.

[0158]The bioavailability of the active protein(s) according to the
invention can also be ameliorated by using conjugation procedures which
increase the half-life of the molecule in the human body, for example
linking the molecule to polyethylenglycol, as described in the PCT Patent
Application WO 92/13095.

[0159]The present invention relates to a method of enhancing immunity in a
patient in need, e.g. a patient suffering from an infectious disease and
cancer, comprising administration of a therapeutically effective amount
of said fragment of IL-32.

[0160]A "therapeutically effective amount" is such that when administered,
said fragment of IL-32 results in enhancement of host defense. The dosage
administered, as single or multiple doses, to an individual may vary
depending upon a variety of factors, including the route of
administration, patient conditions and characteristics (sex, age, body
weight, health, size), extent of symptoms, concurrent treatments,
frequency of treatment and the effect desired. Adjustment and
manipulation of established dosage ranges are well within the ability of
those skilled in the art, as well as in vitro and in vivo methods of
determining the activity said IL-32 fragments.

[0161]While this invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications. This application is intended to cover any variations, uses
or adaptations of the invention following, in general, the principles of
the invention and including such departures from the present disclosure
as come within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended claims.

[0162]All references cited herein, including journal articles or
abstracts, published or unpublished U.S. or foreign patent application,
issued U.S. or foreign patents or any other references, are entirely
incorporated by reference herein, including all data, tables, figures and
text presented in the cited references. Additionally, the entire contents
of the references cited within the references cited herein are also
entirely incorporated by reference.

[0163]Reference to known method steps, conventional methods steps, known
methods or conventional methods is not any way an admission that any
aspect, description or embodiment of the present invention is disclosed,
taught or suggested in the relevant art.

[0164]The foregoing description of the specific embodiments will so fully
reveal the general nature of the invention that others can, by applying
knowledge within the skill of the art (including the contents of the
references cited herein), readily modify and/or adapt for various
application such specific embodiments, without undue experimentation,
without departing from the general concept of the present invention.
Therefore, such adaptations and modifications are intended to be within
the meaning an range of equivalents of the disclosed embodiments, based
on the teaching and guidance presented herein. It is to be understood
that the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by the
skilled artisan in light of the teachings and guidance presented herein,
in combination with the knowledge of one of ordinary skill in the art.

[0165]The present invention will now be described in more detail in the
following non-limiting examples and the accompanying drawings.

EXAMPLES

Example 1

Isolation of an IL-32 Binding Protein from Human Urinary Proteins

[0166]An affinity column of human recombinant IL-32 (hrIL-32) was prepared
in order to isolate IL-32 binding proteins from human urine.

[0167]A recombinant human IL-32α (3 mg) produced in E. coli was
coupled to Affigel-15 (1 ml, BioRad, Richmond Calif.), according to the
manufacturer's instructions and packed into a column. A 1000-fold
concentrate of human crude urinary proteins (500 ml) was loaded onto the
column at a flow rate of 0.25 ml/min. The column was washed with 250 ml
of a solution containing 0.5 M NaCl in phosphate buffered saline (PBS).
Column-bound proteins were eluted with a solution containing 25 mM citric
acid, pH 2.2 and benzamidine (1 mM) and 1 ml fractions were collected and
neutralized immediately by 1 M Na2CO3 (0-70 microliters. For
Elution 3, 70 microliters of neutralizing solution were used). Aliquots
of the fractions eluted from the column (and of the wash fraction) were
resolved by SDS-PAGE (10%) under non-reducing conditions and the protein
bands were visualized by silver staining. A broad band corresponding to a
specific IL-32 binding protein of 28-32 kDa was detected mainly in eluted
fraction 3 (arrow in FIG. 1). This band was not seen in the wash
fraction, which represents crude urinary proteins.

[0168]The results show that a fraction of urinary proteins enriched with
an IL-32 binding protein of 28-32 kDa was obtained by affinity
chromatography with a column of hrIL-32.

Example 2

Identification of IL-32 Binding Protein as Proteinase 3

[0169]The following experiment was carried out in order to identify the
urinary IL-32 binding protein enriched by affinity chromatography with
the hrIL-32 column (Example 1).

[0170]The band from the SDS-PAGE from Example 1, corresponding to 28-32
kDa was excised from the gel and the proteins were electro-eluted and
digested with trypsin. The resulting tryptic digest was subjected to
liquid-chromatography and tandem mass spectrometry (LC-MS/MS). The
sequence of three tryptic peptides was unequivocally determined as being:
LFPDFFTR, VALYVDWIR and LVNVVLGAHNVR. Alignment of the sequence of the
three tryptic peptides and tryptic peptides sequences in the protein
database of the National Center for Biotechnology Information (NCBI) at
the National Institute of Health, Bethesda Md., revealed that the IL-32
binding protein isolated by affinity chromatography is the human
Proteinase-3 (PR-3, SwissProt Accession No P24158). An additional protein
corresponding to human immunoglobulin chain was also identified in the
same protein band but it appears to be a non-specific component.

[0171]That IL-32 binding protein is PR-3 was further confirmed by
N-terminal protein microsequence analysis. The 28-32 kDa protein band of
the SDS-PAGE of elution fraction 3 (Example 1 and FIG. 1) was excised
from the gel, electroeluted onto a PVDF membrane according to the
manufacturer's instruction, and subjected to protein microsequencing on a
Model Applied Biosystems instrument. The resulting N-terminal sequence of
the electroeluted 28-32 kDa protein was identical to that of the
commercially available PR-3 (Athens Research and Technology), IVGG. This
sequence is present in positions 28-31 of pro-PR-3.

[0172]The results show that the urinary IL-32-binding-protein of 28-32
kDa, which was isolated by affinity purification, is PR-3.

[0174]IL-32 (20 μg/ml) in acetate buffer pH 4.6) was immobilized to a
single channel of a BIAcore chip as recommended by the manufacturer
(Amersham Pharmacia, Uppsala Sweden). Aliquots of elution fraction 3 from
the IL-32 affinity column containing urinary PR3, were brought to a
concentration of 10, 20, 30, 40 and 80 nM and analyzed by the BIAcore
system. The binding data gave a kD of 2.65×10-8 M. (FIG. 2A).
The same analysis was done with PR-3 that was inactivated by PMSF (1 mM)
(FIG. 2B). The resulting kD was 7.9×10-8 M. The same analysis
was done with a commercially available human neutrophil derived PR-3
(Athens Research and Technology, Athens Ga., 0.5 μg in 20 μl
gelatin solution) (FIG. 3A). The resulting kD was 1.2×10-8 M.
The binding was repeated with commercial PR-3 that was inactivated by
PMSF (FIG. 3B). The resulting kD was 3.5×10-8 M.

[0175]The chip could not be re-used indicating that IL-32 is being cleaved
from the chip by PR3.

[0176]The results obtained show that binding of IL-32 to PR-3 is not
dependent on the enzymatic activity of the latter, since PR-3 pre
incubated with PMSF still binds to the IL-32 with high affinity.

Example 4

Radioactive Labeling of IL-32

[0177]The preceding example shows that IL-32 is a substrate for PR-3. In
order to explore the catalytic activity of PR-3 on IL-32 degradation,
radioactive labeled IL-32 was prepared as follows.

[0178]IL-32 (15 μg in 60 μl phosphate buffer pH 7.4) was iodinated
using the modification of the Chloramine T method. Briefly, a mixture of
Chloramine T (50 μl, 1 mg/ml in H2O) and 1 mCi [125I]-NaI
(10 μl) was incubated for 20 sec at 4° C. and was added to the
IL-32 preparation for additional 20 sec at 4° C. The reaction was
stopped by the addition of sodium meta bisulfite (50 μl of a stock of
5 mg/ml) and potassium iodide (50 μl of a stock of 5 mg/ml).
Radioactive labeled IL-32 was separated from free iodine on Sephadex G25
column (Pharmacia), which was first equilibrated with 0.25% gelatin in
PBS containing 0.025% sodium azide (i.e. gelatin solution). Six 1 ml
fractions were collected using the gelatin solution. Fractions 3 and 4
contained the peak of the labeled of 125I-IL-32 (specific activity
˜2×105 cpm/ng).

Example 5

Kinetics of Degradation of 125I-IL-32 by Urinary PR-3

[0179]The following experiment was carried out in order to explore the
kinetic of IL-32 degradation by PR-3.

[0180]Affinity purified urinary PR-3 (elution 3 fraction from Example 1,
FIG. 1, 50 μl) was added to 125I-IL-32 (250,000 cpm in 10 μl
of gelatin solution) and incubated at 37° C. for 0, 1, 5, 15, 30,
and 60 min. (FIG. 4). Degradation was stopped by the addition of SDS-PAGE
sample buffer and by boiling for 10 min. Samples containing
125I-IL-32 and PR-3 incubated at different periods of time (0-60
min.) were resolved on 12% SDS-PAGE under reducing conditions. The gel
was dried and autoradiographed. The results summarized on FIG. 4 show the
reduction in the 20 kDa band representing intact IL-32 (FIG. 4, lane 1)
and the increase in the levels of a 16 and 13 kDa IL-32 cleavage products
after a 1-minute incubation with PR-3 (FIG. 4, lane 2). It was found that
the 20 kDa band completely disappeared and the levels of the 16 and 13
kDa IL-32 cleavage products increased after a 5 min. incubation with PR-3
(lane 3) and that the high levels of the 16 and 13 kDa IL-32 fragments
remained stable for up to 60 min (FIG. 4, lanes 3, 4, 6, and 7).

[0181]We found that incubation of IL-32 with PR-3 overnight at 4°
C. resulted in a complete digestion and no product higher than the 10 kDa
marker (the limit of the gel) was observed (data not shown).

Example 6

Kinetics of Cleavage of 125I-IL-32 by Urinary and Commercial PR-3

[0182]The experiment presented in FIG. 5 was carried out essentially as
described in example 5, except that it was limited to up to five-minute
incubation of IL-32 with either urinary or commercial PR-3.

[0183]Urinary PR-3 or commercial PR-3 (Athens Research and Technology)
were incubated with 125I-IL-32 for 0, 1 and 5 min. Digestion was
stopped by the addition of SDS-PAGE sample buffer and by boiling for 10
min. The samples were resolved on SDS-PAGE under reducing conditions. The
gel was dried and autoradiographed. The results summarized on FIG. 5 show
that the commercial PR-3 is more potent than the urinary PR-3, leading to
complete disappearance of the 20 kDa IL-32 band within 1 min (compare
lane 6 with lane 2). This difference is probably due to a different
specific activity of urinary vs. commercial PR3.

[0184]The following experiment was carried out to prove that the
degradation of following incubation with PR-3 is due to the proteolytic
activity of PR-3.

[0185]The experiment presented in FIG. 6 was performed as described in
examples 5 and 6, except that PR-3 was pre-incubated with PMSF (final
concentration of 1 mM, 10 min at 37° C.) prior to the incubation
with 125I-IL-32. PMSF completely abolished the ability of urinary
and commercial PR-3 to process IL-32. In contrast to active PR-3 (lane
1,2 and 9,10 for urinary and commercial PR-3, respectively), no cleavage
products were observed with PMSF pretreated PR-3 incubated with IL-32
(FIG. 6, lanes 3, 4, 7, 8).

Example 8

The Effect of PR-3 in the Activity of IL-32

[0186]The results obtained in the preceding examples show that PR-3 binds
with high affinity to IL-32 and cleaves IL-32 within minutes. The
following experiments were carried out to explore the effect of PR-3 on
the biological activity of IL-32.

[0187]Mouse macrophage Raw 264.7 cell line (American Type Culture
Collection, ATCC) was maintained in RP MI-1640 medium containing 10% FCS.
Bioassays were performed in 96 well plates. Briefly, Raw cells
(5×105/ml, 0.1 ml/per well) were seeded and cultured until
cells adhered to the plate. The medium was removed and the cells were
then stimulated with fresh medium (without FCS) containing different
concentration of IL-32 or IL-32 pre-treated with PR-3 for 5 min at room
temperature in the presence 5 μg/ml of the LPS blocker polymyxin B
(Bedford Laboratories, Bedford, Ohio). The plates were incubated at
37° C. with 5% CO2 for 16-20 h and then the culture
supernatants were collected to measure MIP-2 released by the cell
culture. The results are summarized in FIG. 7 and show that
PR-3-pretreated IL-32 enhances the production of MIP-2 in Raw 264.7 cells
compared to non-pretreated IL-32.

[0188]Similar enhancement of IL-32 activity by pre-treatment with PR-3 was
shown in an experiment carried out with human peripheral blood
mononuclear cells (PBMC). PBMC were prepared as previously described
[Kim, 2005 #36] and seeded at a concentration of 5×105 cells
per well in 96 well plates. The experimental setting of stimulation with
IL-32 and IL-32 pre treated with PR-3 was identical to that described
above for the mouse Raw cells. After stimulation of the human PBMC cells
with either IL-32 or IL-32 pre treated with PR-3, the culture
supernatants were collected to measure human IL-8 released by the cells.
The results are summarized in FIG. 8 and show that stimulation with
PR-3-pre-treated IL-32 enhances the production of IL-8 in human PBMC
cells compared to non-pretreated IL-32.

Example 9

PR-3 Fragment Preparation and Activity

[0189]PR3 (Sigma) was reduced and alkylated prior to cleavage by CNBr.
Reduction and alkylation: DTT (18 μl 50 mM) was added to a PR3 sample
(90 μg, 1 mg/ml) and incubated for 1 hour at 56° C.
Iodoacetamide (60 μl, 100 mM) was added and the mixture was incubated
for 45 min at room temperature in the dark. The reaction was stopped by
DTT (33.6 μl, 50 mM). The sample was dried and reconstituted in 70
μl formic acid. Solid Cyanogen Bromide (CNBr) was added for 48 hrs at
room temperature and in the dark. CNBr desalting was done with C18
Zip-tip according to manufacturer instructions. The peptides mixture was
subjected to mass spectrometry (Maldi) which showed formation of two
peptides.

[0190]Another sample of PR3 is reduced, alkylated and cleaved by CNBr. The
resulting peptides are separated by RP-HPLC (Sigma Discovery BIO Wide
Pore C8 HPLC column, 5 μm particle size, 5 cm×4 mm). Since
mature PR-3 has three methionines, up to four peptides are expected to be
formed by the CNBr cleavage: the N-terminal 1678 Kda peptide, a 549 and a
11712 Kda peptide, and a C-terminal 11187 Kda peptide.

[0191]The affinity of each peptide to IL-32 is determined by BIACORE
compared to the affinity of intact enzymatically active PR3 to IL32 and
to the affinity of intact enzymatically non-active PR3 to IL-32.

[0192]The peptides are then tested for inhibition of the biological
activity of IL-32.